Analysis of antimicrobial coatings using XRF

ABSTRACT

A method of quantifying an antimicrobial coatings using a handheld XRF analyzer is disclosed. The method provides an estimate of the expected level of antimicrobial efficacy for a thin film comprising silicon and/or titanium by obtaining a 14Si or 22Ti peak intensity using XRF spectroscopy and converting the obtained 14Si or 22Ti peak intensity to the expected level of efficacy using a calibration curve. A properly calibrated handheld XRF analyzer allows a user to assess the viability of antimicrobial coatings in the field, such as in a hospital where various fomites may be coated with silane and/or titanium compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/749,343 filed Jan. 22, 2020, entitled “Analysis of AntimicrobialCoatings using XRF.” The '343 application claims priority to and thebenefit of U.S. Provisional Patent Application Ser. No. 62/796,821 filedJan. 25, 2019, entitled “Analysis of Antimicrobial Coatings using XRF,”and U.S. Provisional Patent Application Ser. No. 62/902,102 filed Sep.18, 2019, entitled “Analysis of Antimicrobial Coatings using XRF.” The'343, '821 and '102 applications are incorporated herein by reference intheir entireties for all purposes.

FIELD

The present disclosure generally relates to analytical measurementsusing X-ray fluorescence (XRF) spectrometry, and more specifically tosystems and methods for analyzing antimicrobial coatings on surfacesusing a handheld XRF analyzer.

BACKGROUND

In general, it is inherently difficult to quantify thickness andcomposition of thin films coated on surfaces. For example, a large orpermanently fixed surface (e.g., a countertop) cannot be physicallybrought to an analytical laboratory for surface analysis. In otherexamples, the difference in weight between an uncoated and coatedsurface may be undetectable or statistically unreliable, particularly ifthe surface is considerably heavy in weight and the thin film coating iscorrespondingly miniscule in weight, which is often the situation.

Various analytical methods such as atomic force microscopy (AFM), X-rayphotoelectron spectroscopy (XPS), Auger Electron Spectroscopy (AES),Fourier Transform Infrared Spectroscopy (FTIR), and Raman spectroscopyare useful for thin film quantification and characterization. However,of these methods, only FTIR and Raman are sufficiently portable to betaken into the field to analyze thin film coatings on fixed surfaces.Further, and depending on the substances to be analyzed, some of theseanalytical methods do not have sufficient detection sensitivity forquantitative analysis of thin films. See, for example, Encyclopedia ofMaterials Characterization, Butterworth-Heinemann, Stoneham, MAPublishers, C. Brundle, et al., editors, ISBN-13: 978-0-7506-9168-0.

Various antimicrobial coatings applied to surfaces are in the form ofthin films, so the coatings are often difficult or impossible toquantify and characterize out in the field where the coated surfaces arelocated, such as in hospitals and other institutions. Such films mayhave thicknesses in the several monolayers to micron range, with weightsper unit area of less than about 1 mg/in². For some antimicrobialcoatings (e.g., comprising a silane and/or a titanium species), theweight of the antimicrobial coating per unit of area may relate toresidual antimicrobial efficacy of the coating. As such, knowing howmuch antimicrobial coating has been disposed on a surface, or thatremains on the surface after a period of time and wear, is important inestimating the antimicrobial efficacy of the coating. Further, suchcoatings may be worn off by frequent handling of the surface or fromvarious environmental exposures or cleaning protocols, and thus it isimportant to have a portable quantification method able to track theamount of antimicrobial coating remaining on a surface over time.

In spite of the vast knowledge in thin film analysis, there still existsa need for new portable analytical methods for quantifying antimicrobialcoatings in the field, applicable on a diverse set of surfaces such asplastics and metals, non-destructive, rapid and inexpensive, in order topredict antimicrobial efficacy of a coated surface as the coating isworn off over time. In particular, a portable analytical method forquantifying antimicrobial coatings in a healthcare setting, such as acoating comprising a silane or titanium species, is needed.

SUMMARY

Contaminated surfaces are a critical risk factor for transmittinginfectious disease. Current disinfection products provide short-termantimicrobial action; however, these surfaces can be re-contaminatedwithin hours after cleaning. To address this limitation, long-lastingantimicrobial polymer coatings have been developed, as an adjunct totraditional disinfecting and cleaning protocols. Due to the microscalethickness and transparency of such polymer coatings, confirmation of thepresence of a coating on a surface is difficult with conventionalmethods. To solve this problem, this disclosure provides a novelapproach to measuring durable polymer coatings on glass, plastics,stainless steel and other surfaces to validate their presence andrelative antimicrobial activity.

In various embodiments, a hand-held X-Ray Fluorescence Spectroscopy(XRF) analyzer is utilized to quantitatively evaluate the amount ofantimicrobial polymer coating deposited on test surfaces and remainingafter mechanical abrasion and washing. Additionally, the relationshipbetween the XRF spectra and antimicrobial activity was evaluated using amodified version of an existing sanitization protocol for hard surfacesusing Staphylococcus epidermidis as the test organism. The analyticmethod using XRF is simple, inexpensive, rapid, portable, quantifiable,and provides direct proof of the presence of an antimicrobial coating.

In various embodiments, a method of quantifying antimicrobial silaneand/or titanium thin films using portable X-ray fluorescencespectroscopy (XRF) has now been discovered. In various embodiments, anXRF ₁₄Si and/or ₂₂Ti peak intensity becomes a predictor of antimicrobialefficacy and durability of antimicrobial thin films comprising a silaneand/or a titanium species. In various embodiments, an antimicrobialcoating comprises a silane having a quaternary ammonium chloridesubstituent, allowing for analysis of the coating and prediction ofantimicrobial efficacy by assessing ₁₄Si and/or ₁₇Cl content in thecoating by XRF.

The method comprises using a handheld XRF analyzer for quantifying anamount of silicon atoms (₁₄Si), titanium atoms (₂₂Ti), and/or chlorine(₁₇Cl) atoms present in an antimicrobial thin film coating, and relatingthe quantity of ₁₄Si, ₂₂Ti and/or ₁₇Cl to an expected antimicrobialefficacy of the coating by using a calibration curve. In variousembodiments, a calibration curve allows for interpolation of expectedresidual antimicrobial efficacy of an antimicrobial coating given ameasured photon count of an element known to be present in the coating,such as silicon, titanium or chloride. In various embodiments, acalibration curve of antimicrobial efficacy versus elemental photoncounts is specific to a particular organism, such as a bacteria or virusspecies.

In various embodiments, a method of estimating an expected level ofresidual antimicrobial efficacy for an antimicrobial coating comprisingsilicon and/or titanium comprises: obtaining ₁₄Si or ₂₂Ti photon countsfrom the antimicrobial coating using XRF spectroscopy; and convertingthe obtained ₁₄Si or ₂₂Ti photon counts to the expected level ofresidual antimicrobial efficacy using a calibration curve.

In various embodiments, a method of measuring a thickness of anantimicrobial coating comprising silicon and/or titanium comprises:obtaining ₁₄Si or ₂₂Ti photon counts from the antimicrobial coatingusing XRF spectroscopy; and converting the obtained ₁₄Si or ₂₂Ti photoncounts to the thickness using a calibration curve.

In various embodiments, a method of measuring the weight per unit ofsurface area of an antimicrobial coating comprising silicon and/ortitanium comprises: obtaining ₁₄Si or ₂₂Ti photon counts from theantimicrobial coating using XRF spectroscopy; and converting theobtained ₁₄Si or ₂₂Ti photon counts to the weight per unit of surfacearea using a calibration curve.

In various embodiments, the antimicrobial coating comprises a silaneselected from the group consisting of 3-(trimethoxysilyl) propyldimethyl octadecyl ammonium chloride, 3-(trihydroxysilyl) propyldimethyl octadecyl ammonium chloride, 3-chloropropyltrimethoxysilane,3-chloropropyltriethoxysilane, 3-chloropropylsilanetriol,3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane,3-aminopropylsilanetriol, homopolymers therefrom, and mixtures thereof.In certain aspects, XRF analysis of antimicrobial coatings comprising3-(trimethoxysilyl) propyl dimethyl octadecyl ammonium chloride or3-(trihydroxysilyl) propyl dimethyl octadecyl ammonium chloride maycomprise obtaining ₁₇Cl photon counts, or photon counts from anotherhalide associated with the ammonium substituent if not chloride. Invarious embodiments, the antimicrobial coating comprises nanoparticulateTiO₂.

In various embodiments, the step of obtaining the ₁₄Si or ₂₂Ti photoncounts comprises irradiation of the antimicrobial coating with X-raysemanating from a handheld XRF analyzer and detecting X-ray emissionsfrom the coating.

In various embodiments, the calibration curve comprises an x/y plot ofthe expected level of residual antimicrobial efficacy for a desiredmicroorganism versus the ₁₄Si or ₂₂Ti photon counts.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

The subject matter is pointed out with particularity and claimeddistinctly in the concluding portion of the specification. A morecomplete understanding, however, may best be obtained by referring tothe detailed description and claims when considered in connection withthe following drawing figures:

FIG. 1 shows an XRF spectrum of a thin film comprising an organosilaneand a titanium-sol on an aluminum coupon, before and after coating.Samples were scanned three times and the image shows an overlay of allsix spectra;

FIG. 2 shows ₁₄Si and ₂₂Ti XRF spectra before and after a thin filmcomprising both an organosilane and titanium-sol is applied to glass.The overlaid spectra demonstrate that ₂₂Ti provides a useful measurementof the film on glass, whereas ₁₄Si does not because of the presence ofsilicon atoms in the glass substrate;

FIG. 3 shows ₁₄Si and ₂₂Ti XRF spectra before and after a thin filmcomprising both an organosilane and titanium-sol is applied to whiteHDPE. The spectra demonstrate that ₁₄Si provides a useful measurement ofthe film on the white plastic, whereas ₂₂Ti does not because of thepresence of TiO₂ in the pigmented plastic substrate;

FIG. 4 shows a plot of relative standard deviation (RSD) versus scanningtime demonstrating an increase in the precision of the data withincreasing scanning time;

FIG. 5 shows a plot of silicon photon count versus scanning time, withlinearity demonstrating a constant ratio of silicon photon count toscanning duration;

FIG. 6 shows a plot of silicon photon count at constant current (50 μA)versus voltage varied from about 6 kV to about 40 kV indicatinglinearity particularly in the midrange and the suitability of choosing50 μA current;

FIG. 7 shows a plot of silicon photon count at constant voltage (10 kV)versus current varied from about 5 μA to about 180 μA, indicating nosignificant advantage in data precision by voltage variation and thusthe suitability of 10 mV;

FIG. 8 shows two combined bar charts, with the right bar chart showingscans of 8 mm areas indicated as spots 1-5 (i.e., multiple scanning)spaced apart by about 50-100 cm, and the left bar chart showing severalmeasurements of each spot to improve precision (i.e., averaging);

FIG. 9 shows a plot of silicon photon counts versus average weight forvarious plastics, in order to arrive at a universal calibration curve;

FIG. 10 shows a plot of silicon photon counts versus average weight forvarious metals, in order to arrive at a universal calibration curve;

FIG. 11 shows results when stainless steel coupons were weighed andplaced at different locations in a professional sports facility beforeelectrostatic application of the antimicrobial coating product.Post-treatment XRF analysis results were compared to the net increaseweight of the stainless-steel coupons;

FIG. 12 shows direct XRF analysis of compatible surfaces (low backgroundnoise and homogenous composition) in the field, performed in locationsadjacent to pre-weighed stainless steel reference coupons;

FIG. 13 shows comparison of the amount of antimicrobial polymer coating(in mg/in²) and the XRF silicon photon count resulting in a calibrationcurve having a high degree of linearity (R²=0.9959);

FIG. 14 shows ₁₄Si XRF spectra for a progressively abraded antimicrobialthin film comprising a silane on a stainless-steel coupon. The overlaidXRF spectra demonstrate the usefulness of the XRF method in monitoringthe amount of antimicrobial thin film remaining on a surface as the thinfilm is mechanically abraded;

FIG. 15 sets forth a wearing profile of an antimicrobial coating,wherein treated stainless steel coupons were subjected to differentabrasion conditions on a Gardco™ straight line washability machine usedto represent routine cleanings or frequent handling of the coatedsurface. The plot shows that XRF can be used to detect the wearing ofthe coating caused by routine cleanings;

FIG. 16 shows plots of coating coverage versus number of wear cycles forcoatings subjected to mechanical wear or three different cleaners(VIREX, bleach or OXIVER). The amount of coating remaining wasdetermined by XRF analysis and using the calibration curve; and

FIG. 17 shows a plot of antimicrobial efficacy (log10 reduction) versuscoating weight/in², wherein the coating weights were determined by XRFanalysis.

DETAILED DESCRIPTION

The detailed description of exemplary embodiments refers to theaccompanying drawings, which show exemplary embodiments by way ofillustration and their best mode. While these exemplary embodiments aredescribed in sufficient detail to enable those skilled in the art topractice the invention, it should be understood that other embodimentsmay be realized, and that logical, chemical, and mechanical changes maybe made without departing from the spirit and scope of the inventions.Thus, the detailed description is presented for purposes of illustrationonly and not of limitation. For example, unless otherwise noted, thesteps recited in any of the method or process descriptions may beexecuted in any order and are not necessarily limited to the orderpresented. Furthermore, any reference to singular includes pluralembodiments, and any reference to more than one component or step mayinclude a singular embodiment or step. Also, any reference to attached,fixed, connected or the like may include permanent, removable,temporary, partial, full and/or any other possible attachment option.Additionally, any reference to without contact (or similar phrases) mayalso include reduced contact or minimal contact.

In various embodiments, a method of surface analysis using X-rayfluorescence (XRF) spectroscopy is disclosed. In various aspects, an XRFsurface analysis technique is used to analyze an antimicrobial coatingpreviously applied to a surface of an object. In various aspects, XRFsurface analysis provides both weight and thickness of an antimicrobialcoating. In various examples, XRF surface analysis provides a way toverify the antimicrobial efficacy such a coating will provide afterrepeated handling, mechanical and chemical abrasion, or from washing ofthe coating. In various embodiments, the surface material may compriseFormica®, stainless steel or other metals and alloys, glass, or variousplastics. The objects may comprise fomites in a healthcare setting, suchas countertops, sinks, faucets, door handles, bed rails, arms of chairs,tables, food trays, and the like.

In various embodiments, a method of measuring a thickness of anantimicrobial coating comprising a silane and/or a titanium speciescomprises obtaining a ₁₄Si and/or ₂₂Ti peak intensity from the coatingusing XRF spectroscopy, and converting the obtained ₁₄Si and/or ₂₂Tipeak intensity to the thickness of the antimicrobial thin film using acalibration curve.

In various embodiments, a method of measuring a weight per unit ofsurface area of an antimicrobial coating comprising a silane and/or atitanium species comprises obtaining a ₁₄Si and/or ₂₂Ti peak intensityfrom the coating using XRF spectroscopy and converting the obtained ₁₄Siand/or ₂₂Ti peak intensity to the weight per unit of surface area of theantimicrobial coating using a calibration curve.

In various embodiments, a method of predicting a level of residualantimicrobial efficacy for a silane and/or titanium speciesantimicrobial coating comprises obtaining a ₁₄Si and/or ₂₂Ti peakintensity from the silane and/or titanium coating using XRF spectroscopyand converting the obtained ₁₄Si and/or ₂₂Ti peak intensity to thepredicted level of residual antimicrobial efficacy using a calibrationcurve.

Definitions and Interpretations:

As used herein, the term “XRF” refers to X-ray fluorescence spectrometryand specifically to an X-ray fluorescence surface analysis methodwherein a sample is irradiated with X-rays having a certain energy froma controlled X-ray tube to excite particular atoms present in thesample. When the atoms are struck by the X-rays they release fluorescentX-rays having energy equal to the difference in energy between quantumstates of the electrons of the atoms. The energy of the emitted photonsidentifies the element, and the peak height and/or intensity of thefluorescence correlates to the concentration of the element in thesample. What results from XRF analysis is a spectrum of peak intensities(units of CPS, i.e., counts/second, or magnitudes 10² CPS, 10³ CPS,etc., as needed) versus wavelength (e.g., in nm). The wavelength rangeis typically from about 0.01 nm to about 10 nm, corresponding toenergies of 125 keV to 0.125 keV, so that elemental information isobtained for elements in the range from magnesium to uranium. For themethods disclosed herein, photon counts of ₁₄Si or ₂₂Ti atoms is thencorrelated to the quantity of silicon or titanium in a coating analyzed.

XRF is a relative measurement, meaning that calibration standards in thewavelength (element) range of interest are required to transpose thepeak intensities, such as for ₁₄Si and/or ₂₂Ti intensities, intoabsolute elemental compositions and thicknesses. The quantification isperformed using empirical calibration functions. Quantification withfundamental parameters allows XRF to be used as an absolute technique.For an explanation of these concepts, see P. van De Weijer, et al.,“Elemental Analysis of Thin Layers by X-Rays,” Philips J. Res., 47,247-262, 1993.

As used herein, the term “XRF analyzer” refers to an instrument capableof performing XRF spectroscopy. An analyzer will typically comprise anX-ray source, a pre-amp detector, a digital signal processor and a CPU.Of interest herein are handheld XRF analyzers that combine all thenecessary components into a single housing shaped like a power tool andhaving a LED screen to read information. Handheld XRF analyzers find usein screening products coming off production lines, sorting scrap metalsand researching antiquities in the field. A handheld XRF surfaceanalyzer is utilized herein to analyze antimicrobial coatings that mayhave been applied to various surfaces in a healthcare facility, andelsewhere where portability of the analyzer is important.

As used herein, the term “antimicrobial” is used generally to indicateat least some level of microbe kill by a liquid composition or a driedcoating present on a portion of a surface. For example, antimicrobialmay be used to indicate a biostatic efficacy, sanitizing level (3-log,or 99.9%) reduction in at least one organism, or a disinfection level(5-log, or 99.999%) reduction in at least one organism, or sterilization(no detectable organisms). Microbes, or microorganisms, may include anyspecies of bacteria, virus, mold, yeast, or spore. Of interest hereinare bacterial organisms found in healthcare settings and otherinstitutions, such as, but not limited to, E. coli, S. aureus, MRSA, S.epidermidis, S. saprophyticus, S. agalactiae, S. pneumoniae, S.pyogenes, S. typhi, S. typhimurium, P. aeruginosa, M pneumoniae, Mjeprae, M tuberculosis, and M. ulcerans, and various viral species suchas HIV, hepatitis A, B, C, D, E, influenza, SARS coronavirus, and H1N1.

The terms “residual antimicrobial,” “residual self-sanitizing,” and“self-decontaminating surface” are used interchangeably to indicate aphysical property of a coating on a surface of a hard, inanimate object,namely that the coating is capable of maintaining antimicrobial efficacyover a certain period of time under certain conditions once the surfaceis coated with an antimicrobial coating composition and the compositiondried on the surface. A coating on a surface may maintain residualantimicrobial efficacy indefinitely, or the coating may eventually “wearout,” such as when mechanically abraded or washed from the surface,losing its residual antimicrobial efficacy. An antimicrobial coatingcomposition (e.g., a liquid solution) may function as a contactsanitizer, disinfectant, or sterilant when first applied to a surface,killing the organisms already present on the surface, and also have theability to leave behind a residual antimicrobial coating on the surfaceonce the composition is dried or cured thereon, to keep inactivating newmicroorganisms that contact the dried coating. In various embodiments,coating compositions may not be antimicrobial until dried or cured on asurface but are still referred to as antimicrobial coating compositionsherein because of their ability to produce a residual antimicrobialeffect on the surface, and for the sake of brevity. The residualantimicrobial effect exhibited by the antimicrobial coatings is notlimited by a particular mechanism of action, and no such theories areproffered. For example, an antimicrobial effect measured for a coatingon a surface may be the result of intracellular mutations, inhibition ofcertain cellular processes, rupture of a cell wall, immobilization andthus prevention of transfer or detection when swabbing, or a nondescriptinactivation of the organism. Other antimicrobial effects may includeinhibiting the reproduction of an organism or inhibiting the organism'sability to accumulate into biofilms. In other embodiments, anantimicrobial effect may be a stasis such that organisms cannotproliferate to the point of reaching a pathogenic level on the coatedsurface.

As used herein, the term “antimicrobial coating composition” or“residual self-sanitizing coating composition” refers to a chemicalcomposition, primarily liquid, comprising at least one chemical species,which is used to produce a residual self-sanitizing antimicrobialcoating on a surface after the composition is applied to a surface of anobject and then allowed to dry. However, the term is extended to includea composition that may be applied sequentially (e.g., over or under) orcontemporaneously with the application of an antimicrobial coatingcomposition comprising a recognizable antimicrobial active. In variousembodiments, this precoating or overcoating may act as an adherent or asealant to assist in bonding the residual antimicrobial coating to thesurface, improve durability of the overall coating, and/or to provide acatalytic effect or some sort of potentiation or synergy with theresidual antimicrobial coating comprising an antimicrobial active. Forsimplicity, each one of multiple compositions used sequentially orcontemporaneously to produce an overall residual antimicrobial coatingon a surface is referred to as an “antimicrobial coating composition,”even if one or more of the compositions used for coating has noidentifiable antimicrobial active or where the active agent isuncertain. For example, a coating composition comprising a silane may befirst applied to a surface of an object and dried, followed by a coatingcomposition comprising a titanium species, which is applied to thesurface and dried.

An antimicrobial coating composition herein may comprise a neat, 100%active chemical species or may be a solution or suspension of a singlechemical species in a solvent such as water or an alcohol (methanol,ethanol, iso-propanol, etc.). In other embodiments, a composition maycomprise a complex mixture of chemical substances, some of which maychemically react (hydrolyze, self-condense, etc.) within the compositionto produce identifiable or unidentifiable reaction products. Forexample, a monomeric chemical species in an antimicrobial coatingcomposition may partially or fully polymerize while in solution prior toa coating process using that composition. In various embodiments,chemical constituents within an antimicrobial coating composition maychemically react on the surface that the composition is applied to, suchas while the composition is drying and concentrating on the surface.Antimicrobial coating compositions for use in various embodiments mayfurther comprise any number and combination of inert excipients, such asfor example, solvents, surfactants, emulsifiers, stabilizers,thickeners, free-radical initiators, catalysts, etc. Exemplaryantimicrobial coating compositions that leave behind a residualself-sanitizing coating on a surface, and that may benefit from XRFsurface analysis per the methods disclosed herein, include, but are notlimited to, solutions comprising and organosilane selected from thegroup consisting of 3-(trimethoxysilyl) propyl dimethyl octadecylammonium chloride, 3-(trihydroxysilyl) propyl dimethyl octadecylammonium chloride, 3-chloropropyltrimethoxysilane,3-chloropropyltriethoxysilane, 3-chloropropylsilanetriol,3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane,3-aminopropylsilanetriol, and mixtures thereof. Any combination of thesesilanes, optionally including an organic amine such as triethanolamine,may be used in an aqueous or alcoholic solution amenable to spray or dipcoating of surfaces. In various embodiments, an antimicrobial coatingcomposition is sprayed onto a surface, and in some instances,electrostatically sprayed.

In various embodiments, antimicrobial coatings that may benefit fromanalysis using the XRF surface analysis methods herein, are disclosed inthe following patent references, each assigned to Allied Bioscience,Inc, and each of which is incorporated herein by reference in itsentirety for all purposes: U.S. Pat. Nos. 10,463,046; 10,456,493;10,421,870; 10,420,342; 10,258,046; 10,238,114; 10,194,664; 10,182,570;10,040,952; 10,040,097; 9,963,596; 9,918,475; 9,856,360; 9,855,584;9,757,769; and 9,528,009; and U.S. patent application Ser. No.16/591,785 filed Oct. 3, 2019.

As used herein, the shorthand notation “2015” indicates an antimicrobialcoating composition or a dried coating obtained therefrom comprising3-(trimethoxysilyl) propyl dimethyl octadecyl ammonium chloride and/or3-(trihydroxysilyl) propyl dimethyl octadecyl ammonium chloride. Theshorthand “2020” indicates an antimicrobial coating composition or adried coating obtained therefrom comprising3-chloropropyltrimethoxysilane, 3-chloropropyltriethoxysilane, and/or3-chloropropylsilanetriol. The shorthand “2030” indicates anantimicrobial coating composition or a dried coating obtained therefromcomprising 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane,and/or 3-aminopropylsilanetriol. A “T” placed after any of thesedesignations, e.g., “2015-T,” indicates a coating obtained by both the2015 silane antimicrobial composition and a titanium speciescomposition. Such a combined coating may be obtained by simultaneousapplication, or stepwise application, in either order, or a 2015composition and a titanium species composition.

In various embodiments, an antimicrobial thin film for analysis hereincomprises a titanium coating, either alone or in conjunction with asilane antimicrobial thin film. In some instances, the titanium coatingmay act as a sealant or adherent for a silane antimicrobial thin film,such as applied underneath or overtop the silane coating on a surface.In various aspects, a titanium coating herein can be quantified by XRFspectrometry, specifically by the ₂₂Ti peak intensities in an XRFspectrum. In various aspects, a titanium coating may be more simplyreferred to herein as an antimicrobial thin film because it may be usedin concert with a silane antimicrobial thin film. A titaniumantimicrobial thin film may comprise a titanium(IV) species, asdescribed below.

As used herein, the term “titanium (IV) species” refers to any chemicalcompound comprising at least one tetravalent titanium atom, regardlessif monomeric, dimeric, trimeric, or polymeric. Non-limiting examplesinclude titanium (IV) oxide (TiO₂) in any form, other Ti(IV) species,(e.g., TiCl₄, Ti-(O-i-C₃H₇)₄ or any other Ti(IV) alkoxide, phenoxide orhalide). Various forms of TiO₂ for use herein include, but are notlimited to, rutile, anatase, brookite, hollandite-like,ramsdellite-like, α-PbO₂-like, baddeleyite-like form, orthorhombicTiO₂-OI, cubic, and/or cotunnite-like forms. The most common crystallineforms are anatase, brookite and rutile. In various examples, Ti(IV)species for use herein comprise Ti nanoparticles. Further, Ti(IV)species for use herein include “titanyl-oxide moieties,” which is abroad term defined herein to include any and all Ti compounds andmixtures known to form TiO₂ thin films, or at least suspected as able toform TiO₂ thin films, such as via the sol-gel process. A titanyl sol-gelis a precursor in the preparation of TiO₂ thin films. For example, amixture of Ti(OC₄H₉)₄, ethanol, water, and diethanolamine, in a1:26.5:1:1 molar ratio, has been disclosed as forming a TiO₂ film (seeJ. Yu, et al., Materials Chemistry and Physics, vol. 69, pp 25-29(2001)). This reference further discloses that whether or not the filmis photocatalytic depends, inter alia, on the curing conditions for thesol-gel after surface application, e.g., using high temperatures. Inanother non-limiting example, a sol-gel route to mesoporous andnanocrystalline anatase thin layers begins with acidic hydrolysis oftitanium isopropoxide, (see F. Bosc, Chem. Mater., 15(12), pp 2463-2468,(2003)).

In certain examples, titanyl-oxide moieties for use herein comprise acolloidal suspension of from about 0.5 wt. % to about 50 wt. % TiO₂ inwater. In other examples, titanyl-oxide moieties comprise an aqueousmixture of Ti—(O-i-C₃H₇)₄ usable to create a thin film of TiO2 via thesol-gel process. Such compositions may also comprise an organic solvent,such as an alcohol like n-propanol or n-butanol, a surfactant, or anacid catalyst. In the sol-gel process, TiO₂ is prepared by hydrolysis,condensation and polycondensation of a titanium alkoxide, such asTi—(O-i-C₃H₇)₄ or TiCl₄. A TiO₂ sol-gel composition, when coated onto aportion of a surface, provides a thin film TiO₂ coating on the portionof the surface.

In various embodiments, titanyl-oxide moieties comprise Ti(OR³)₄,wherein R³ is alkyl, substituted alkyl, aryl, or substituted aryl, andwherein the four separate R³ groups are identical or different. Examplesof Ti(OR³)₄include, but are not limited to, titanium tetramethoxide,titanium tetraethoxide, titanium methoxide triethoxide, titaniumtetra-n-propoxide, titanium tetra-i-propoxide, and titaniumtetraphenoxide. Depending on the physical properties of the titanium(IV) species, the compound may be used neat (e.g., Ti—(O-i-C₃H₇)₄) ordissolved in an alcohol or other organic solvent(s), such as thecorresponding alcohol, where feasible, (methanol, ethanol, i-propanol,etc.). Thus, titanyl-oxide moieties may in some instances comprise asolution of Ti—(O-i-C₃H₇)₄ in isopropanol or some other alcohol.

In various embodiments, titanyl-oxide moieties comprise Ti(OR³)₄,wherein R³ is alkyl, substituted alkyl, aryl, or substituted aryl. Incertain aspects, titanyl-oxide moieties may further comprise a solventselected from the group consisting of water, alkanols, diols, triols,chlorinated organic solvents, ethers, amines, esters, ketones,aldehydes, lactones, phenolics, and mixtures thereof. In certainexamples, a solvent is selected from, but not limited to, water,methanol, ethanol, n-propanol, i-propanol, ethylene glycol,1,2-propanediol, 1,3-propanediol, glycerin, methylene chloride,trichloromethane, carbon tetrachloride, ethylene glycol monoalkyl ether,ethylene glycol dialkylether, propylene glycol monoalkyl ether,propylene glycol dialkyl ether, ethylene glycol monophenyl ether,ethylene glycol diphenyl ether, propylene glycol monophenyl ether,propylene glycol diphenyl ether, diethylether, tetrahydrofuran,pyridine, triethanolamine, diethanolamine, triethylamine, ethylacetate,acetone, furfural, and N-methyl-2-pyrrolidone, and combinations thereof.In various examples, titanyl-oxide moieties consist essentially ofTi—(O-i-C₃H₇)₄. Other examples include Ti—(O-i-C₃H₇)₄ and an alcohol,and a composition comprising Ti—(O-i-C₃H₇)₄ and iso-propanol.

In various examples, titanyl-oxide moieties for use herein comprise anaqueous solution of peroxotitanium acid and peroxo-modified anatase sol,which is disclosed in the literature as a room temperature route to TiO₂thin films, (see Ichinose, H., et al., Journal of Sol-Gel Science andTechnology, September 2001, Volume 22, Issue 1-2, pp 33-40, andIchinose, H., et al., J. Ceramic Soc. Japan, Volume 104, Issue 8, pp715-718 (1996)).

In various examples, the titanyl-oxide moieties for use herein is asol-gel that comprises about 0.5 wt. % peroxotitanium acid and about 0.5wt. % peroxo-modified anatase sol, remainder water. A non-limitingexample of a titanyl-oxide moieties composition for use herein comprises0.85 wt. % of a mixture of peroxotitanium acid and peroxo-modifiedanatase sol (titanium oxide (IV)), remainder water. In various examples,a titanyl-oxides moieties composition comprises 0.8-0.9 wt. % of amixture of titanium oxide (IV) and peroxotitanium acid, with theremainder, i.e., 99.1-99.2 wt. %, water. In various embodiments, thissol-gel mixture may be referred to as “0.85 wt. % aqueous peroxotitaniumacid and peroxo-modified anatase sol.”

This titanyl sol-gel, or other sol-gels prepared by other processes asdiscussed, may be coated onto a surface by itself, or in combinationwith an antimicrobial silane coating. In an example where the surfacecomprised a borosilicate glass slide, AFM imaging (50 μm² scan area)revealed a 0.85 wt. % aqueous peroxotitanium acid and peroxo-modifiedanatase sol coating, when dry, to have an average roughness of 25.76 nm.In an example where the surface comprised mica, AFM imaging (1 μm² scanarea) revealed a 0.85 wt. % aqueous peroxotitanium acid andperoxo-modified anatase sol coating, when dry, provides an averageparticle size of 30 nm. Although not wishing to be bound by anyparticular theory, these particles may comprise, at least in part,nanoparticulate TiO₂.

As used herein, the term “organosilane,” or more simply, “silane,”refers to silicon-containing organic chemicals, as opposed to inorganicforms of silicon, such as SiO₂ or water glass species (Na₂SiO₃, and thelike). An organosilane is typically a molecule including carbon andsilicon atoms but may also include any other heteroatoms such as oxygen,nitrogen, or sulfur. Organosilane compounds for use in variousembodiments herein may be chemically reactive or inert, and may bemonomeric, dimeric, trimeric, tetrameric, or polymeric. Organosilanemonomers for use in various embodiments may be chemically reactive inthat they at least partially hydrolyze or self-polymerize or formvarious adducts and/or polymers with other chemical species in acomposition or on a surface. Exemplary organosilanes for measurementherein include, but are not limited to, organosilanes having threereactive groups bonded to silicon and one non-hydrolyzable group bondedto silicon. Such organosilanes for use herein include, but are notlimited to, 3-(trimethoxysilyl) propyl dimethyl octadecyl ammoniumchloride, 3-(trihydroxysilyl) propyl dimethyl octadecyl ammoniumchloride, 3-chloropropyltrimethoxysilane, 3-chloropropyltriethoxysilane,3-chloropropylsilanetriol, 3-aminopropyltrimethoxysilane,3-aminopropyltriethoxysilane, 3-aminopropylsilanetriol, and mixturesthereof, and any of their self-condensation products (e.g., homopolymershaving any chain length distribution).

As used herein, the term “thin film” or “coating” (used interchangeablyherein) takes on its ordinary meaning in surface chemistry. For purposesherein, a “thin film” is a layer, having from atomic-scale tomicron-scale thickness, disposed on a substrate. The substrate hereinmay be more simply referred to as a surface, although it should beunderstood the surface is part of a larger substrate. The systems andmethods herein include the quantification of silane and/or titanium thinfilms disposed on surfaces. The surfaces for use herein are not limitedin any way, and may include such materials as plastics, glass, metals,ceramics, wood, paper, and composites such as Formica®. Formica® isunderstood to be a decorative laminate product composed of severallayers of kraft paper impregnated with a melamine thermosetting resin,topped with a decorative layer protected by melamine. The laminate iscompressed and cured with heat. Thus, a silane or titanium coating on aFormica® surface herein is understood to be a silane or titanium thinfilm disposed on a paper and melamine resin laminate. In many ways, aFormica® surface is a plastic surface, wherein the plastic comprisesmelamine. An “antimicrobial thin film” of interest herein exhibits atleast some level of residual antimicrobial efficacy, and the presence ofsuch a thin film on a surface is an indication the surface will provideat least some reduction in organism count when the surface is inoculatedwith an organism.

In various embodiments, an antimicrobial thin film herein comprisesdimethyloctadecyl 3-(trimethoxysilyl)propyl ammonium chloride. In water,this material likely exists as the silanetriol, i.e3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride. Invarious examples, an antimicrobial coating composition is made bydiluting dimethyloctadecyl 3-(trimethoxysilyl)propyl ammonium chloridein water. A non-limiting commercial source of dimethyloctadecyl3-(trimethoxysilyl)propyl ammonium chloride is Sigma-Aldrich, in theform of a 42 wt. % actives solution in methanol. In other examples, anantimicrobial coating composition is made by diluting3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride in water.In various examples, the coating is applied to a surface and dried toform the antimicrobial thin film.

In various embodiments, an antimicrobial thin film herein comprises3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride.3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride iscommercially available from INDUSCO, Inc. in 0.5 wt. %, 0.75 wt. %, 1.5wt. %, 5.0 wt. % and 71.20 wt. % aqueous solutions, under the trade nameBioShield®. The 5 wt. % solution of 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride is also available from INDUSCO, Inc.under the trade name ProShield® 5000D, having EPA Reg. No. 53053-8. Thelabel for ProShield® 5000D further lists the active ingredient as“octadecylaminodimethyltrihydroxysilyl propyl ammonium chloride,” (whichis perhaps an incorrect name for 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride). Another supplier of 5 wt. %aqueous octadecylaminodimethyltrihydroxysilyl propyl ammonium chlorideis Gelest, Inc., 11 East Steel Rd., Morrisville, Pa, 19067 USA. TheGelest MSDS discloses this product as containing 94-96 wt. % water and4-6 wt. % octadecylaminodimethyltrihydroxysilyl propyl ammoniumchloride. These various commercial materials may be used “as is” ordiluted with water and/or other solvents as necessary to obtain thedesired finished weight percent concentration of quaternary silane,e.g., for example, 0.75 wt. %. In various aspects, the composition isapplied to a surface and dried to form an antimicrobial thin film.

In various embodiments, an antimicrobial thin film herein comprises amixture of dimethyloctadecyl 3-(trimethoxysilyl)propyl ammonium chlorideand at least one amine.

In various embodiments, an antimicrobial thin film herein comprises atleast one amine having structure R⁹R¹⁰R¹¹N, wherein R⁹, R¹⁰, and R¹¹ areindependently H, alkyl, substituted alkyl, aryl, substituted aryl orcyclic. In certain examples, an organic amine comprises diethanolamineor triethanolamine.

In certain aspects, an antimicrobial thin film herein comprises asecondary or tertiary amine. In certain examples, an antimicrobial thinfilm herein comprises may comprise dimethyloctadecyl3-(trimethoxysilyl)propyl ammonium chloride and triethanolamine ordiethanolamine. In certain examples, an antimicrobial coatingcomposition comprises a mixture of from about 0.5 wt. % to about 1.0 wt.% dimethyloctadecyl 3-(trimethoxysilyl)propyl ammonium chloride. Invarious examples, an antimicrobial coating composition further comprisesfrom about 0.01 wt. % to about 0.10 wt. % triethanolamine. The coatingcomposition is applied to a surface and dried to form an antimicrobialthin film.

In various embodiments, an antimicrobial coating composition comprisesabout 0.75 wt. % dimethyloctadecyl 3-(trimethoxysilyl)propyl ammoniumchloride; about 0.045 wt. % triethanolamine; and about 99.205 wt. %water. The coating composition is applied to a surface and dried to forman antimicrobial thin film.

In various embodiments, an antimicrobial thin film comprises3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride and atleast one amine. In certain aspects, the amine may be a secondary ortertiary amine. For example, an antimicrobial thin film may comprise3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride andtriethanolamine or diethanolamine. In certain examples, an antimicrobialcoating composition comprises an aqueous mixture of from about 0.5 wt. %to about 1.0 wt. % 3-(trihydroxysilyl)propyl dimethyloctadecyl ammoniumchloride. In various examples, an antimicrobial coating compositioncomprises from about 0.01 wt. % to about 0.10 wt. % triethanolamine. Theantimicrobial coating composition is applied to a surface and dried toform an antimicrobial thin film for analysis herein.

In various embodiments, an antimicrobial coating composition comprisesabout 0.75 wt. % 3-(trihydroxysilyl)propyl dimethyloctadecyl ammoniumchloride; about 0.045 wt. % triethanolamine; and about 99.205 wt. %water. The antimicrobial coating composition is applied to a surface anddried to form an antimicrobial thin film for analysis herein.

In various embodiments, an antimicrobial coating composition comprisesan aqueous mixture of dimethyloctadecyl 3-(trimethoxysilyl)propylammonium chloride, at least one amine, and3-chloropropyltrimethoxysilane and/or 3-chloropropylsilanetriol. Somecommercially sourced dimethyloctadecyl 3-(trimethoxysilyl)propylammonium chloride or 3-(trihydroxysilyl)propyl dimethyloctadecylammonium chloride may contain small amounts of3-chloropropyltrimethoxysilane. A commercial synthesis ofdimethyloctadecyl 3-(trimethoxysilyl)propyl ammonium chloride comprisesthe S_(N)2 reaction between dimethyloctadecylamine and3-chloropropyltrimethoxysilane. In some embodiments, an excess of3-chloropropyltrimethoxysilane may be used to drive this reaction tocompletion. If not separated out from the reaction product mixture, theunreacted 3-chloropropyltrimethoxysilane may remain in the sample ofdimethyloctadecyl 3-(trimethoxysilyl)propyl ammonium chloride. Forexample, a commercial source of 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride may be disclosed to comprise 5.0 wt.% 3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride and 1.0wt. % 3-chloropropyltrimethoxysilane. The antimicrobial coatingcomposition is applied to a surface and dried to form an antimicrobialthin film for analysis herein.

In various embodiments, an antimicrobial coating composition comprises3-chloropropyltrimethoxysilane and dimethyloctadecyl3-(trimethoxysilyl)propyl ammonium chloride and/or3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride. Invarious examples, 3-chloropropyltrimethoxysilane may be added to asolution of dimethyloctadecyl 3-(trimethoxysilyl)propyl ammoniumchloride and/or 3-(trihydroxysilyl)propyl dimethyloctadecyl ammoniumchloride known to not comprise any 3-chloropropyltrimethoxysilane as abyproduct. In other examples, additional 3-chloropropyltrimethoxysilanemay be added to a solution of dimethyloctadecyl3-(trimethoxysilyl)propyl ammonium chloride and/or3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride known toinclude some residual 3-chloropropyltrimethoxysilane as a byproduct. Theantimicrobial coating composition is applied to a surface and dried toform an antimicrobial thin film for analysis herein.

In various embodiments, an antimicrobial coating composition comprisesfrom about 0.5 wt. % to about 1.0 wt. % dimethyloctadecyl3-(trimethoxysilyl)propyl ammonium chloride; from about 0.05 to about0.5 wt. % 3-chloropropyltrimethoxysilane and from about 0.01 wt. % toabout 0.10 wt. % triethanolamine, with the remainder being water. Theantimicrobial coating composition is applied to a surface and dried toform an antimicrobial thin film for analysis herein.

In various embodiments, an antimicrobial coating composition comprisesfrom about 0.5 wt. % to about 1.0 wt. % 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride; from about 0.05 to about 0.5 wt. %3-chloropropyltrimethoxysilane and from about 0.01 wt. % to about 0.10wt. % triethanolamine, with the remainder being water. The antimicrobialcoating composition is applied to a surface and dried to form anantimicrobial thin film for analysis herein.

In various embodiments, an antimicrobial coating composition comprisesabout 0.75 wt. % dimethyloctadecyl 3-(trimethoxysilyl)propyl ammoniumchloride; about 0.06 wt. % 3-chloropropyltrimethoxysilane; about 0.045wt. % triethanolamine; and about 99.145 wt. % water. When applied to aportion of a surface and allowed to dry, this composition provides abiostatic coating. In various embodiments, the treated surface comprisesa mixture of quaternary and 3-chloropropyl surface bound silanes.

In various embodiments, an antimicrobial coating composition comprisesabout 0.75 wt. % 3-(trihydroxysilyl)propyl dimethyloctadecyl ammoniumchloride; about 0.06 wt. % 3-chloropropyltrimethoxysilane; about 0.045wt. % triethanolamine; and about 99.145 wt. % water. When applied to aportion of a surface and allowed to dry, this composition provides abiostatic coating. In various embodiments, the treated surface comprisesa mixture of quaternary and 3-chloropropyl surface bound silanes.

In various embodiments, an antimicrobial coating composition comprisesabout 0.75 wt. % dimethyloctadecyl 3-(trimethoxysilyl)propyl ammoniumchloride; about 0.12 wt. % 3-chloropropyltrimethoxysilane; about 0.045wt. % triethanolamine; and about 99.085 wt. % water. When applied to aportion of a surface and allowed to dry, this composition provides abiostatic coating. In various embodiments, the treated surface comprisesa mixture of quaternary and 3-chloropropyl surface bound silanes.

In various embodiments, an antimicrobial coating composition comprisesabout 0.75 wt. % 3-(trihydroxysilyl)propyl dimethyloctadecyl ammoniumchloride; about 0.12 wt. % 3-chloropropyltrimethoxysilane; about 0.045wt. % triethanolamine; and about 99.085 wt. % water. When applied to aportion of a surface and allowed to dry, this composition provides abiostatic coating. In various embodiments, the treated surface comprisesa mixture of quaternary and 3-chloropropyl surface bound silanes.

In various embodiments, an antimicrobial coating composition comprisesabout 0.75 wt. % dimethyloctadecyl 3-(trimethoxysilyl)propyl ammoniumchloride; about 0.26 wt. % 3-chloropropyltrimethoxysilane; about 0.045wt. % triethanolamine; and about 98.945 wt. % water. When applied to aportion of a surface and allowed to dry, this composition provides abiostatic coating. In various embodiments, the treated surface comprisesa mixture of quaternary and 3-chloropropyl surface bound silanes.

In various embodiments, an antimicrobial coating composition comprisesabout 0.75 wt. % 3-(trihydroxysilyl)propyl dimethyloctadecyl ammoniumchloride; about 0.26 wt. % 3-chloropropyltrimethoxysilane; about 0.045wt. % triethanolamine; and about 98.945 wt. % water. When applied to aportion of a surface and allowed to dry, this composition provides abiostatic coating. In various embodiments, the treated surface comprisesa mixture of quaternary and 3-chloropropyl surface bound silanes.

In various embodiments, a surface is treated with an antimicrobialcoating composition comprising: about 0.75 wt. %3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride; about0.045 wt. % triethanolamine; and about 99.205 wt. % water and allowed tovisibly dry. In one non-limiting example, borosilicate glass slides werepositioned vertically, and electrostatic spray coated from a distance ofabout 5 to 6 feet with this composition. The treated slides were allowedto dry at room temperature overnight. AFM imaging (49 μm×74 μm scanarea) revealed the silane/triethanolamine coating to have an averagethickness of 22.12±3.28 nm, and an average roughness of 19.85±5.62 nm.

In various embodiments, an antimicrobial coating is prepared on aportion of a surface by a method comprising: (1) coating the portion ofthe surface with an aqueous mixture comprising 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride; triethanolamine; and water; and (2)coating the same portion of the surface with aqueous peroxotitanium acidand peroxo-modified anatase sol, in either order (i.e., (1) then (2), or(2) then (1)). Not wishing to be bound by any particular theory, theperoxotitanium acid and peroxo-modified anatase sol coating may assistin adhering the 3-(trihydroxysilyl)propyl dimethyloctadecyl ammoniumchloride to the portion of the surface, and/or may increase thehydrophilicity of the portion of the surface previously made hydrophobicby surface bound 3-(trihydroxysilyl)propyl dimethyloctadecyl ammoniumchloride. Either of these phenomena are possible regardless of the orderof disposition on the portion of the surface. In various examples, XRFspectrometry is used to determine the amount of ₁₄Si and/or ₂₂Ti atomsin the thin film coating.

In various embodiments, an antimicrobial coating is prepared on asurface by a method comprising: (1) coating a portion of the surfacewith an aqueous mixture comprising: 0.75 wt. % 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride; 0.045 wt. % triethanolamine; and99.205 wt. % water; and (2) subsequently coating the portion of thesurface with 0.85 wt. % aqueous peroxotitanium acid and peroxo-modifiedanatase sol. In a non-limiting example, borosilicate glass slides werepositioned vertically, and electrostatic spray coated from a distance ofabout 5 to 6 feet with the aqueous 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride and triethanolamine solution andallowed to dry about 3 to 5 minutes, after which time the 0.85 wt. %aqueous peroxotitanium acid and peroxo-modified anatase sol waselectrostatic spray coated overtop of the organosilane from about 5 to 6feet distance. The treated slides were left to dry at room temperatureovernight. AFM imaging (50 μm² scan area) revealed that the coatingresulting from this two-step sequential surface treatment had an averagethickness of 51.79±17.98 nm, and an average roughness of 35.90±9.43 nm.In various examples, XRF spectrometry is used to determine the amount of₁₄Si and/or ₂₂Ti atoms in the thin film coating.

The method of stepwise surface treatment may be performed in theopposite order. For example, a portion of a surface may be coated firstwith an aqueous solution of peroxotitanium acid and peroxo-modifiedanatase sol, and then the same portion of the surface subsequentlycoated with an aqueous solution of 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride and triethanolamine such that theorganosilane is theoretically overtop the titanyl species. For eitherorder of application, the first coating may be allowed to partly dry orcompletely dry prior to the subsequent coating. In other aspects, thefirst treatment may be applied, and while still wet, followed by thesecond treatment, and then the combined treatments are allowed to dry.Throughout this disclosure, stepwise treatment of a surface is meant totarget approximately the same portion of the surface with successivecompositions. In some instances, a second treatment may liquefy acoating applied first and dissolve those components that were firstdried on the surface. In various examples, XRF spectrometry is used todetermine the amount of ₁₄Si and/or ₂₂Ti atoms in the thin film coating,which can be correlated to the thickness, the weight per unit of surfacearea, and the antimicrobial efficacy of the thin film coating.

A unique situation arises when analyzing an organosilane antimicrobialcoating on a glass surface using XRF. In this case, the substrate, beingglass, is composed of high levels of silicon. Thus, detection of adifference in the level of ₁₄Si peak intensities by XRF before and aftertreatment of a glass surface is not possible. In these instances, oneoption is to use a titanium coating in conjunction with the silanecoating, such as a sealant coating, and to measure the amount of ₂₂Tiatoms in the coating rather than the ₁₄Si levels. Since an XRF spectrumtaken across a range of wavelengths can show both the ₁₄Si and the ₂₂Tipeaks in the spectrum, the analysis becomes straightforward, and theoperator can just pay attention to the ₂₂Ti peak knowing that the ₁₄Sipeak is unreliable since its intensity is due in part to the glasssubstrate. In specific embodiments where the antimicrobial coating onglass comprises a silane having a quaternary ammonium chloridesubstituent, ₁₇Cl photon counts may be obtained instead of ₂₂Ti counts.

EXAMPLES

Antimicrobial Coating Composition, Test Coupons and Spray CoatingMethods

The antimicrobial coating composition for the examples (abbreviated as2015) consisted essentially of: 0.75 wt. % 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride, 0.12 wt. %3-chloropropyltrimethoxysilane, 0.045 wt. % triethanolamine, remainderwater, with the weight percentages based on the total weight of thecomposition.

A series of 1″ by 1″ annealed coupons (glass, metals or plastics, asindicated) were coated with the 2015 product. To maximize the uniformityof coating the test coupons in a laboratory setting, a robotic sliderwas equipped with an electrostatic sprayer and a group of couponsarranged in a tight pattern were sprayed in different passes of thesprayer. The coating procedure comprised spraying the antimicrobialcoating composition as a fine mist from the electrostatic spray gun at adistance of about 3 feet onto the test coupons and allowing the surfacesto dry at room temperature overnight. A plurality of test coupons weresprayed at the same time with the coupons grouped together, typically ina 4 coupon×15 coupon rectangular grid. In various tests, each row of 15coupons may be assigned to a group of replicates, Group 1, Group 2,Group 3 and Group 4. An application “cycle” refers to one sprayapplication as per above. Multiple cycles indicate repeated applicationsof the 2015 composition, which will necessarily build up a heavier filmon the test coupons. This process of accumulating material by repeatingthe application cycle can be monitored by weight measurement or by XRF.Spraying of surfaces in the field, including examples where test couponswere placed on objects outside of a laboratory setting, are describedbelow with the associated example.

The Handheld XRF Device and Software

The hand-held X-Ray Fluorescence Spectroscopy (XRF) analyzer used was aBruker Tracer 5i spectrometer, having a range from ₉F to ₉₂U. The systemuses a 20 mm² silicon drift detector with <140 eV @ 250,000 cps Mn Kαresolution for optimum light element analysis. Rhodium thin window X-raytube is the excitation source and the instrument is equipped with an 8μm beryllium detector window. The acquired data were analyzed withBruker's ARTAXControl XRF software. In various embodiments, the BrukerTitan S1 XRF analyzer can be used, having a range of ₁₂ Mg to ₉₂U. For abrief discussion on interpreting XRF data, see A. Shugar, “Peaking YourInterest: An introductory explanation of how to interpret XRF data,”WAAC Newsletter, (Western Association of Art Conservation), 31(3), 8-10,Sep. 2009,(http://cool.conservation-us.org/waac/wn/wn31/wn31-3/index.html).

XRF Optimization and Developing a Universal Calibration Curve

Developing an XRF method useful for analyzing antimicrobial coatings onsurfaces includes, but is not limited to, optimization of scanning time,voltage and current, and addressing background (BG) signals fromdifferent surfaces under the coating. Ultimately, a universalcalibration curve is developed that can convert measured photon countreadings (e.g., ₁₄Si, ₁₇Cl, and/or ₂₂Ti) from an antimicrobial coatinginto coating thickness, coating weight/unit of surface area, and apredicted level of antimicrobial efficacy expected from theantimicrobial coating. The measuring of ₁₄Si, ₁₇Cl, and/or ₂₂Ti photoncounts is not meant to be limiting, as other elements known to presentin an antimicrobial coating may be analyzed by XRF in accordance to themethods herein, depending for example on the nature of the substratebeneath the coating that may result in unmanageable background signals.In examples where an antimicrobial coating comprises a quaternaryammonium salt, a silane comprising a quaternary ammonium salt, or evensimply a salt, various halogens may be quantified by XRF and the photoncounts interpolated into expected antimicrobial efficacy of the coating.In some instances, a salt may be added to a coating composition as amarker, even if not having any antimicrobial efficacy. In variousembodiments, a halogen is quantified by XRF, including ₁₇Cl, ₃₅Br and₅₃I. For example, an antimicrobial coating may comprise3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium chloride,3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium bromide, or3-(trihydroxysilyl)propyl dimethyloctadecyl ammonium iodide, in whichcase XRF may be used to quantify ₁₇Cl, ₃₅Br or ₅₃I, respectively, inplace of, or in addition to, quantification of the ₁₄Si present.

Example 1: Proof of Concept

Aluminum coupons were sequentially coated with the 2015 organosilaneantimicrobial coating composition above followed by a titanium-solcomposition consisting essentially of 0.85 wt. % of a mixture ofperoxotitanium acid and peroxo-modified anatase sol, remainder water,according to the spray application protocol above. The XRF spectra ofthe resulting thin film are shown in FIG. 1, wherein “before” indicatesthe coupon prior to coating and “after” indicates the coupon aftercoating, As shown in the spectra of FIG. 1, the peak intensities for thesilicon, titanium and aluminum elements can be seen both before andafter coating. For this example, each sample was scanned three times,with the spectra in FIG. 1 representing an overlay of all six spectra.As shown in FIG. 1, both the ₁₄Si and ₂₂Ti peaks are not interfered withby the aluminum, but instead are clearly usable as shown by the starkdifference in ₁₄Si and ₂₂Ti peak intensities for uncoated versus coatedaluminum. As demonstrated, the ₁₄Si or ₂₂Ti peak intensities can be usedto determine the amount of thin film coating on the surface, and theamount remaining on the surface after environmental wear, to provide anestimate of the expected residual antimicrobial efficacy from the thinfilm coating.

Example 2: Background Considerations—Antimicrobial Coating on SilicaGlass Surfaces

Glass coupons were sequentially coated with the 2015 antimicrobialcoating composition and a titanium-sol composition per above. The XRFspectra of the resulting thin film are shown in FIG. 2, wherein peakintensities for the silicon and titanium elements can be seen bothbefore and after coating. This example demonstrates that the ₂₂Ti peakintensities find use for assessing the antimicrobial coating containingtitanium on the glass surface, whereas the ₁₄Si peak intensities are notuseful because of the interference from the silicon atoms in the glasssubstrate. From the spectra on the left side of the figure, one can seethat there is no discernable difference in the ₁₄Si peak intensitiesbetween an uncoated glass slide and a coated glass slide because of thehigh ₁₄Si peak intensity from the underlying glass substrate. From thespectra on the right side of the figure, one can see that there is adiscernable difference in the ₂₂Ti peak intensities between an uncoatedglass slide and a coated glass slide because there is no titanium in theglass substrate to interfere with the assessment of the coating thatcontains a titanium species.

Example 3: Background Considerations—Antimicrobial Coating on WhitePigmented Plastic Surfaces

White pigmented high density polyethylene (HDPE) coupons weresequentially coated with the 2015 antimicrobial coating composition andthe titanium-sol composition per above. The XRF spectra of the resultingthin film are shown in FIG. 3, wherein peak intensities for the siliconand titanium elements can be seen both before and after coating. Thisexample demonstrates that the ₁₄Si peak intensities find use forassessing the antimicrobial coating on the HDPE surface, whereas the₂₂Ti peak intensities are not of any use because of the interferencefrom the TiO₂ pigment present in the white plastic substrate. From thespectra on the left side of the figure, one can see that there is aneasily discernable difference in the ₁₄Si peak intensities between anuncoated white HDPE slide and a coated white HDPE slide because of nosilicon atoms in the underlying plastic substrate. From the spectra onthe right side of the figure, one can see that there is no discernabledifference in the ₂₂Ti peak intensities between an uncoated white HDPEslide and a coated white HDPE slide because of the presence of TiO₂pigment in the white plastic substrate.

Example 4: Scanning Duration Time

A stainless-steel coupon was scanned at 3 different spots with scanningdurations varying from 10 seconds to 120 seconds and the corresponding₁₄Si photon counts were obtained. The relative standard deviation (RSD)can be an indicator of the closeness of the acquired data. As shown inFIG. 4, increasing scanning time of the XRF analyzer improves theprecision of the data. Due to practical reasons, a compromise is made sothat an operator can analyze a coating in a particular location in areasonable amount of time. For example, 10 seconds scanning durationseems to be the most reasonable choice for a large sample size.Evidently, the ratio of silicon photon count to the scanning durationappears to be constant, as shown by the linearity of the ₁₄Si photoncount versus scanning time graph in FIG. 5. This observation is criticalregarding normalization of the data acquired with different scanningdurations.

Example 5: Effects of Current and Voltage in the Analyzer

The available range for voltage depends on the chosen value for currentand vice versa. In this experiment, voltage and scanning time were keptconstant at 10 kV and 10 seconds respectively and the scanning was of ablank (uncoated) glass test coupon. The current was increased from 5 to180 μA. The results shown in FIG. 6 indicate a generally linear relationbetween current and photon count, but particularly so in the mid-rangeof the plot. From the substantially linear portion seen in FIG. 6, fromabout 50 μA to about 100 μA, 50 μA appears to be the lowest suitablecurrent for differentiating silicon peaks without overheating theinstrument with unnecessarily high current.

As shown in FIG. 7, varying the voltage from about 6 kV to about 40 kVwhile maintaining a constant current of about 50 μA showed a spike inthe photon count around 20 kV, however there was no significantadvantage seen in the RSD. Therefore, 10 kV was believed optimal and waschosen for the rest of the experiments.

Example 6: Sampling Size and Averaging

Ideally, XRF scanning should provide assessment of the coverage of aacross a surface. However, the measuring area for a handheld XRFanalyzer is about an 8 mm diameter spot. To assess advantages torepeating measurements of the same area or different areas and theadvantages to averaging, a large plastic surface without any coating wasscanned by XRF. The uncoated surface was selected for its consistency inbackground materials. As shown by the left five bars in the bar graph ofFIG. 8 (spots 1 . . . 5), each of five distinct 8 mm spots, spaced apartfrom each other on the plastic surface by about 50 cm to about 100 cm,were scanned 5 separate times. The error bars shown on the left fivebars indicates the variations between the 5 scans for each spot.Alternatively, and as shown by the right five bars in the bar graph ofFIG. 8, each bar represents the scanning of one spot only once. The fivebars collectively show each of 5 spots scanned once. The error barsshown at the top of the five right bars indicate variation between thespots.

Example 7: Compatibility of Various Surfaces Underneath AntimicrobialCoatings

To be able to analyze antimicrobial coatings such as 2015 on surfacesother than stainless steel, the present method was tested forcompatibility with various materials, both metallic and non-metallic. Avariety of substrate materials found in healthcare facilities werecoated with the 2015 silane antimicrobial coating and analyzed with theXRF analyzer. The plastics that were coated and the coatingssubsequently analyzed included acrylonitrile butadiene styrene (ABS),high density polyethylene (HDPE), faux leather (vinyl, likely comprisinga mixture of materials), polyoxymethylene (acetal), polycarbonate (PC),polypropylene (PP), polyamide (nylon), polytetrafluoroethylene (PTFE),and polymethylmethacrylate (acrylate). The metals that were coated andthe coatings subsequently analyzed included 304 stainless steel, 316stainless steel, 464 brass, 260 brass, 1100 aluminum, 2024 aluminum,zinc, copper and titanium. After optimization and subtraction of thebackground signal (as indicated by the table entries “-BG,” meaning“minus the background BG signal”), calibration curves were obtained foreach material type. As seen in FIG. 9 (variety of plastics) and FIG. 10(variety of metals), the similarity between the slopes of ₁₄Si photoncount versus mg/in² for the silane coating on a variety of differentmaterials suggests that a universal calibration curve is achievable.However, as seen in Example 2 for glass, analysis of a silaneantimicrobial coating on substrates bearing a high silicon content(e.g., glass, ceramic tiles, marbles, silicon rubber) remainsproblematic.

Example 8: Actual Weight Versus Predicted Weight of Coatings on VariousSurfaces

In this example, information was collected from actual spraying in thefield, as proof the XRF method and the calibration curve are applicableto a real-life environment where various objects are sprayed in order tocoat with an antimicrobial coating. For the first part of this example,pre-weighed 1″×1″ stainless steel coupons were placed on each of avariety of surfaces on familiar objects. The purpose of using standardtest coupons on various real-life surfaces was to remove any variabilitycaused by differences in materials and their associated backgroundsignals. The surfaces were coated with the 2015 antimicrobial coatingcomposition, impinging on the test coupons placed thereon, and allowedto dry. The test coupons were collected from the various surfaces andthe handheld XRF analyzer was used to measure ₁₄Si photon counts. Thesecounts were converted to a predicted weight of coating using thecalibration curve of ₁₄Si photon counts versus weight in mg/in². The barchart of FIG. 11 shows comparisons between actual weight (i.e., measuredweight) and predicted coating weight (i.e., determined from XRF readingsand interpolation on the calibration curve).

For the second part of this example, the surfaces in close proximity tothe previously placed test coupons were scanned using XRF. The testcoupons placed on the surfaces provided the actual weight/in², whereasthe XRF photon counts of the surfaces around where the test coupons wereplaced provided predicted weight/in². The ₁₄Si photon counts thusobtained were converted to a predicted coating weight in mg/in² usingthe calibration curve (“direct scan”). The corresponding test coupon ofthe same or similar material was analytically weighed to determine theweight of coating (“coupons”). The results are provided in the bar graphof FIG. 12. This example shows the utility of XRF for assessment ofcoating weight in the field where the objects cannot be physically movedand/or directly weighed.

Example 9: Calibration

Comparison of the amount of the 2015 antimicrobial coating (in mg/in²)and the XRF silicon photon count provided the calibration curve setforth in FIG. 13 having a high degree of linearity (R²=0.9959)especially for surfaces coated with less than about 1 mg/in²antimicrobial coating. Reliability in the method seems to trail offabove about 4.0 mg/in² of antimicrobial coating, which is far morecoating than one would practically apply to surfaces in an institution.Typical coating weights using the 2015 antimicrobial coating compositionand an electrostatic sprayer for coating hospital furnishings is about0.12 mg/in².

The data to construct the calibration curve of FIG. 13 was obtained bycoating grids of 4 coupons×15 coupons, using the robotic sprayer, withthe coupons being 1″×1″ brushed 304 stainless steel. Each column of 4coupons were removed from the grid after 2, 4, 6, 8, 10, 12, 16, 20, 24,30, 36, 42, 50, 60 and 70 passes of the sprayer. Each of the fourcoupons in a column represented four replicates. The experiment wasrepeated twice, i.e., two grids of 4 coupons×15 coupons, and the twosets of test results averaged. Having two sets of results showed themethod to be reproducible. Recognizing that the calibration curve is forSi-containing coatings on stainless steel, the Y-intercept, whichdepends on the background ₁₄Si counts, can be adjusted for differentsubstrates. This aspect can be programmed into software customized forcoating analysis in healthcare settings where there is likely both metaland plastic surfaces requiring antimicrobial coatings.

Example 10: Mechanical Abrasion of Coatings and Monitoring Loss ofCoating

1. Proof of concept: Stainless-steel coupons were sequentially coatedwith the 2015 organosilane antimicrobial coating composition and thetitanium-sol composition per the above examples. After an initial XRFspectrum was taken of the coating, the coated slide was inserted into anabrasion tester and abraded per the abrasion protocol described below.An XRF spectrum was recorded after each abrasion cycle, and all thespectra overlaid to produce the plot shown in FIG. 14. As shown in FIG.14, the ₁₄Si peak intensities provide a way to quantify theantimicrobial thin film as it is worn off the surface through mechanicalabrasion. As explained below, mechanical abrasion in a laboratorysetting mimics the frequent handling of surfaces. Modifications of thelaboratory mechanical abrasion protocol can mimic the repeated cleaningof surfaces. Further examples of wear testing are disclosed hereinbelow.

(i) Mechanical Abrasion Testing of Antimicrobial Surfaces

Antimicrobial coatings can be subjected to mechanical abrasion and XRFcan be used to track a decrease in the amount of antimicrobial coatingand predict the corresponding decrease in antimicrobial efficacy. Weardata are indicative of the durability of a coating and relate to howwell an antimicrobial coating can withstand frequent handling or otherenvironmental insult. For example, wear testing in the laboratory canmimic the repeated handling of a door knob. Or with cleaning solutionsincluded in the laboratory wear testing, the laboratory method can mimicthe repeated cleaning of a surface that was previously coated with anantimicrobial coating. An existing EPA Protocol may be used to generatethe wear data in the laboratory. In certain instances, the EPA protocolmay be modified, such as to add cleaners that might be used in aninstitution where various surfaces are coated with antimicrobialcoatings.

EPA Protocol #01-1A, entitled “Protocol for Residual Self-SanitizingActivity of Dried Chemical Residues on Hard, Non-Porous Surfaces,” is astandard test method used for testing the durability of an antimicrobialcoating on a hard surface. The test method utilizes an in-line abrasionmachine commonly used in assessing the cleaning ability of detergents.However, instead of a standard soiled tile being positioned in themachine to be scrubbed, test coupons previously coated with anantimicrobial coating per the above spray method are positioned in themachine. The back-and-forth cycling of a weighted scrubber (a weighted“boat” with a cloth or sponge) simulates natural wearing of theantimicrobial coating, such as the wear the surface may experience whenfrequently handled. In variations of the test protocol, the cloth in theweighted boat may be moist to simulate the handling of surfaces with amoist hand, or wet with a cleaner to simulate cleaning of the surfaces.For moistening, the cloth was placed 75 cm away from the cleaner orwater sprayer nozzle and was sprayed for 1 second. In various examples,correlations can be made to handling of environmental surfaces, e.g., adoorknob. At various wear cycles, coupons may be weighed for weight lossor inoculated with a test organism.

The abrasion tester suggested in the EPA protocol is a GardCo™Washability and Wear Tester, Model D10V, Cat. No. #WA-2153, from thePaul N. Gardner Co., Inc., Pompano Beach, Fla., which is the machineused herein. Variables in the protocol include the weight of the boat,the material wrapped around the boat (e.g., a cloth wiper), the moisturelevel on the wiper, the speed of the oscillations, and the number ofcycles, in addition to the type of antimicrobial coating on the testcoupons, the test coupon material, and the arrangement of coated couponsin the machine (i.e., the pattern).

(ii) Abrasion Protocol

The action of abrasion of an antimicrobial thin film may be monitored byweight loss. 1″×1″ stainless steel test coupons were used, each couponweighed before and after coating, and before and after abrasion testing.

The wear testing is performed in replicates of two.

TexWipe® cotton wipers (VWR# TWTX309) were used with TexWipe® FoamWipe™wipers (VWR# TWTX704) as a liner on the weighted boat.

The weight of the boat was adjusted to a total weight of 1.0 kg byadding auxiliary weights.

Using the GardCo™ Washability machine, a cycle refers to 2 passes of theweighted boat, there and back. Abrasion speed was set to “2.5,” whichequated to about 4-6 seconds per cycle.

The cotton wiper and foam liner were arranged in the weighted boat. Thewiper was sprayed at a distance of 75 cm±1 cm with deionized water for 1second using a Preval® Sprayer to moisturize the wiper. Abrasion testingwas performed immediately after moisturizing the wiper.

The TexWipe® cotton wiper was replaced after each abrasion cycle.

Test coupons subjected to 10 cycles (10×) or 30 cycles (30×) are thenmeasured for percent weight loss or inoculated with a test organism tomeasure residual antimicrobial efficacy.

2. Basic abrasion to model frequent handling of coated surfaces:Abrasion of 2015 antimicrobial coatings prepared from the 2015composition above on stainless steel coupons was followed by measuring₁₄Si photon counts with the XRF analyzer. In this example, the boat ofthe washability machine was either dry or wetted with water per theprotocol above. The results are shown in FIG. 15 as plots of ₁₄Si photoncounts versus number of wear cycles for the three abrasion conditions—noweight on the boat, 500 g weight added to the boat, and 500 g weightadded to the boat with the boat wrapped in a wipe wetted with water.

3. Abrasion to model washing of coated surfaces with cleaners: Anothermajor objective of the method herein was to provide a tool formonitoring the wearing of the coating over time and to provide anestimation of the appropriate retreatment time, i.e., the time when auser should recoat the surface with antimicrobial coating. To betterunderstand the wearing profile of the coating, the Gardco™ washabilitymachine and the wear protocol was used to represent the effects ofroutine cleanings on an antimicrobial coating. It is reasonable toassume that surfaces in a healthcare setting, even if coated with anantimicrobial coating, will still be periodically washed in accordancewith standard hygiene protocols.

To simulate wear and cleaning of surfaces, a series of 1″ by 1″stainless steel coupons were coated with the 2015 antimicrobial coatingcomposition as per above. The weighted boat on the Gardco™ machine wasequipped with a cotton cloth. In series of tests, the cloth was sprayedwith different common disinfectants (2 sprays from about 75 cmdistance). Following each wearing cycle (each cycle=one back and oneforward movement/pass of the boat) the samples were analyzed with thehandheld XRF instrument. Using the previously obtained calibrationcurve, the coating coverage was predicted and plotted against the numberof wearing cycles. The results suggest that the upper loose layers ofantimicrobial coating wear off the steel surface with the first fewcycles, and then the abrasion rate of the coating slows downsignificantly. Such observation supports the use of XRF as a method tomonitor antimicrobial coating coverage and durability of the coatingsover periods of time, including washing of the coated surfaces. Asevident from FIG. 16, the effect of disinfectants on the removal of theantimicrobial coating is minimal.

Example 11: Antimicrobial Efficacy of Coatings

The relationship between 14Si photon counts from XRF spectra andresidual antimicrobial activity of a coating was evaluated using amodified version of an existing sanitization protocol (ASTM-E1153) forhard surfaces using Staphylococcus epidermidis (ATCC 12228) as the testorganism. Treated and untreated control coupons were inoculated with1×10⁶ cfu/mL S. epidermidis and held at room temperature for a contacttime of 2 hours. After completion of the contact time, carriers wereimmediately placed into 20 mL of D/E neutralizer broth (HardyDiagnostics) and vortexed for 30 seconds. Dilutions of each coupon wereplated in duplicate using the pour plate method with Tryptic Soy Agar(TSA, BD Biosciences). Plates were incubated at 37° C. for 36-48 hours;after incubation, plates with 30-300 colonies were counted and thecfu/carrier was calculated. The cfu/carrier values were used todetermine the log₁₀ reduction for each coupon versus the untreated (2hour) control. A total of 4 coupons were tested per weight from twoindependent experiments.

To produce a curve of efficacy versus weight of antimicrobial coating,the ₁₄Si photon counts were converted to the corresponding weight ofcoating in mg/in² using the calibration curve. The results of theefficacy experiments are shown in FIG. 17 as a plot of log₁₀ reductionof Staphylococcus epidermidis (ATCC 12228) versus coating weight inmg/in². the relationship between XRF values and antimicrobial efficacywere found to be well-correlated with a logarithmic trend (R²=0.9308).

The trend reveals a sharp increase in antimicrobial activity afterformation of initial coating layers (low coating weight), and theaddition of more layers does not have an additive impact on efficacy.This observation is aligned with various possible mechanisms of actionof such coatings, where only the exposed outermost layer is in contactwith organisms. Increasing the thickness of the coating does notincrease the surface area with a linear rate, which explains theobserved plateau in the rate of antimicrobial activity.

This final calibration curve allows one to take the XRF analyzer intothe field, such as into a hospital, where surfaces have been coated withthe antimicrobial coating, and instantly determine the expectedantimicrobial efficacy remaining on a coated surface. A determination ofexpected antimicrobial efficacy is more important than just athickness/weight determination since the expected antimicrobial efficacyis also dependent on the organisms that might be inoculated onto acoated surface, such as by touching, and not just the amount ofantimicrobial coating on the surface. As a hypothetical example, thesame remaining weight/in² of silane or silane/titanium coating on asurface may provide a 5-log kill of E. coli but only a 3-log kill of S.epidermidis. Software programming on the XRF analyzer may incorporate acalibration curve obtained for each organism such that the user of theXRF analyzer need only select the organism, take an XRF ₁₄Si photoncount reading from the coating on the surface, and look at a displayscreen on the analyzer or on a supporting computer to see theantimicrobial efficacy expected for the coating for this chosenpathogen. In this way, the staff in the hospital can quickly determinewhich surfaces to recoat with antimicrobial coating composition.

These findings show that the use of a handheld XRF analyzer provides arapid and cost-effective method for assessing the presence and efficacyof antimicrobial coatings on various substrate surfaces.

In the detailed description, references to “various embodiments”, “oneembodiment”, “an embodiment”, “an example embodiment”, etc., indicatethat the embodiment described may include a particular feature,structure, or characteristic, but every embodiment may not necessarilyinclude the particular feature, structure, or characteristic. Moreover,such phrases are not necessarily referring to the same embodiment.Further, when a particular feature, structure, or characteristic isdescribed in connection with an embodiment, it is submitted that it iswithin the knowledge of one skilled in the art to affect such feature,structure, or characteristic in connection with other embodimentswhether or not explicitly described. After reading the description, itwill be apparent to one skilled in the relevant art(s) how to implementthe disclosure in alternative embodiments.

Benefits, other advantages, and solutions to problems have beendescribed herein with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any elements that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as critical, required, or essentialfeatures or elements of the disclosure. The scope of the disclosure isaccordingly to be limited by nothing other than the appended claims, inwhich reference to an element in the singular is not intended to mean“one and only one” unless explicitly so stated, but rather “one ormore.” Moreover, where a phrase similar to ‘at least one of A, B, and C’or ‘at least one of A, B, or C’ is used in the claims or specification,it is intended that the phrase be interpreted to mean that A alone maybe present in an embodiment, B alone may be present in an embodiment, Calone may be present in an embodiment, or that any combination of theelements A, B and C may be present in a single embodiment; for example,A and B, A and C, B and C, or A and B and C.

All structural, chemical, and functional equivalents to the elements ofthe above-described various embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Moreover, itis not necessary for an apparatus or component of an apparatus, ormethod in using an apparatus to address each and every problem sought tobe solved by the present disclosure, for it to be encompassed by thepresent claims. Furthermore, no element, component, or method step inthe present disclosure is intended to be dedicated to the publicregardless of whether the element, component, or method step isexplicitly recited in the claims. No claim element is intended to invoke35 U.S.C. 112(f) unless the element is expressly recited using thephrase “means for.” As used herein, the terms “comprises”, “comprising”,or any other variation thereof, are intended to cover a non-exclusiveinclusion, such that a chemical, chemical composition, process, method,article, or apparatus that comprises a list of elements does not includeonly those elements but may include other elements not expressly listedor inherent to such chemical, chemical composition, process, method,article, or apparatus.

We claim:
 1. A method of estimating an expected level of residualantimicrobial efficacy for an antimicrobial coating comprising anorganosilane having a quaternary ammonium chloride substituent, themethod comprising: obtaining ₁₇Cl photon counts from the antimicrobialcoating using XRF spectroscopy; and converting the obtained ₁₇Cl photoncounts to the expected level of residual antimicrobial efficacy using acalibration curve.
 2. The method of claim 1, wherein the organosilanehaving a quaternary ammonium chloride substituent is selected from thegroup consisting of 3-(trimethoxysilyl) propyl dimethyl octadecylammonium chloride, 3-(trihydroxysilyl) propyl dimethyl octadecylammonium chloride, homopolymers therefrom, and mixtures thereof.
 3. Themethod of claim 1, wherein the antimicrobial coating further comprisesat least one of 3-chloropropyltrimethoxysilane,3-thioropropyltriethoxysilane, 3-chloropropylsilanetriol,3-aminopropyltrimethoxysilane, 3-aminopropyltdethoxysilane, or3-aminopropylsilanetriol.
 4. The method of claim 1, wherein theantimicrobial coating further comprises an organic amine.
 5. The methodof claim 1, wherein the step of obtaining the ₁₇Cl photon countscomprises irradiation of the antimicrobial coating with X-rays emanatingfrom a handheld XRF analyzer and detecting X-ray emissions from thecoating.
 6. The method of claim 1, wherein the calibration curvecomprises an x/y plot of the expected level of residual antimicrobialefficacy for a desired microorganism versus the ₁₇Cl photon counts.
 7. Amethod of measuring a thickness of an antimicrobial coating comprisingan organosilane having a quaternary ammonium chloride substituent, themethod comprising: obtaining initial ₁₇Cl photon counts from theantimicrobial coating using XRF spectroscopy; subjecting theantimicrobial coating to mechanical abrasion; obtaining ₁₇Cl photoncounts from the antimicrobial coating after mechanical abrasion usingXRF spectroscopy; and converting the obtained ₁₇Cl photon counts to thethickness of the antimicrobial coating after mechanical abrasion using acalibration curve.
 8. The method of claim 7, wherein the organosilanehaving a quaternary ammonium chloride substituent is selected from thegroup consisting of 3-(trimethoxysilyl) propyl dimethyl octadecylammonium chloride, 3-(trihydroxysilyl) propyl dimethyl octadecylammonium chloride, homopolymers therefrom, and mixtures thereof.
 9. Themethod of claim 7, wherein the antimicrobial coating further comprisesat least one of 3-chloropropyltrimethoxysilane,3-chloropropyltriethoxysilane, 3-chloropropylsilanetriol,3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, or3-aminopropylsilanetriol.
 10. The method of claim 7, wherein theantimicrobial coating further comprises an organic amine.
 11. The methodof claim 7, wherein the step of obtaining the ₁₇Cl photon countscomprises irradiation of the antimicrobial coating with X-rays emanatingfrom a handheld XRF analyzer and detecting X-ray emissions from thecoating.
 12. The method of claim 7, wherein the calibration curvecomprises an x/y plot of antimicrobial coating thickness versus ₁₇Clphoton counts.
 13. A method of measuring a weight per unit of surfacearea of an antimicrobial coating comprising an organosilane having aquaternary ammonium chloride substituent, the method comprising:obtaining initial ₁₇Cl photon counts from the antimicrobial coatingusing XRF spectroscopy; subjecting the antimicrobial coating tomechanical abrasion; obtaining ₁₇Cl photon counts from the antimicrobialcoating after mechanical abrasion using XRF spectroscopy; and convertingthe obtained ₁₇Cl photon counts to the weight per unit of surface areaof the antimicrobial coating after mechanical abrasion using acalibration curve.
 14. The method of claim 13, wherein the organosilanehaving a quaternary ammonium chloride substituent is selected from thegroup consisting of 3-(trimethoxysilyl) propyl dimethyl octadecylammonium chloride, 3-(trihydroxysilyl) propyl dimethyl octadecylammonium chloride, homopolymers therefrom, and mixtures thereof.
 15. Themethod of claim 13, wherein the antimicrobial coating further comprisesat least one of 3-chloropropyltrimethoxysilane,3-chloropropyltriethoxysilane, 3-chloropropylsilanetriol,3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, or3-aminopropylsilanetriol.
 16. The method of claim 13, wherein theantimicrobial coating further comprises an organic amine.
 17. The methodof claim 13, wherein the step of obtaining the ₁₇Cl photon countscomprises irradiation of the antimicrobial coating with X-rays emanatingfrom a handheld XRF analyzer and detecting X-ray emissions from thecoating.
 18. The method of claim 13, wherein the calibration curvecomprises an x/y plot of weight per unit of surface area of theantimicrobial coating versus ₁₇Cl photon counts.